Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.

The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers.

The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.

SET-domain proteins are methyltransferases that add methyl groups to lysine (K) residues of histone tails, which function as marks activating or repressing transcription. SET-domain proteins can be divided into evolutionarily conserved classes in animals and plants. Mutation of the ASH1 HOMOLOG 2 (ASHH2) gene of the model plant Arabidopsis has severe pleiotropic effects on growth, development and fertility, in that it results in homoetic changes of floral organ identity, affects male and female gametogenesis, and especially the development of the reproductive organs. On the female side, close to 80 percent of the mature ovules lack embryo sac. On the male side, anthers in part develop without pollen sacs (locules) and in developed locules the tapetum layer, which provides material to the pollen wall, is distorted. Together this results in a more that 75 percent reduction in mature functional pollen grains. Transcriptional profiling was used to identify changes in gene expression in ashh2 mutant inflorescences. Genes involved in determination of floral organ identity, embryo sac development and anther/pollen development were found down-regulated. On such genes ChIP detected reduction of H3K36me3 but not H3K4me3 or H3K36me2. Our study indicates that ASHH2 is a major global H3K36 trimethyltransferase in Arabidopsis.

SET-domain proteins add methyl groups to lysine (K) residues of histone tails, which may function as marks activating or repressing transcription. The ASH1 HOMOLOG 2 (ASHH2) protein of Arabidopsis thaliana groups with Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a histone H3K36 methyltransferase, and has been implicated as a histone H3K4 or H3K36 methyltransferase. ashh2 mutants display pleiotropic developmental defects, including early flowering. Here we focus on the role of ASHH2 in plant reproduction, of homeotic changes in floral organ identity and specific effects on the development of the reproductive organs. On the female side, close to 80% of the mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs and where present show specific defects in the tapetum layer. As a result, the number of functional pollen per anther was reduced by up to ~90%. Transcriptional profiling identified more than 600 down-regulated genes in ashh2 mutant inflorescences, including genes involved in determination of floral organ identity, embryo sac development and anther/pollen development. Currently, there is a discrepancy in the literature on the primary substrate of ASHH2 methylation. We observed a reduction of H3K36 trimethylation (me3) but not H3K4me3 or H3K36me2 in chromatin from selected down-regulated genes. Thus, our analysis strongly suggests that ASHH2 works via H3K36 trimethylation in the regulation of genes essential in reproductive development.

The more than 30 SET-domain proteins of Arabidopsis can be classified in evolutionarily conserved subgroups that methylate different lysine residues on histone tails (1).The ASH1 HOMOLOG 2 (ASHH2) protein is similar to the Drosophila transcriptional activator ASH1, and the histone H3K36 methyltransferase Set2 from yeast. ashh2 mutants display pleiotropic developmental defects, including early flowering (2,3). We have focused on the role of ASHH2 in plant reproduction, of homeotic changes in floral organ identity and specific effects on the development of the reproductive organs. On the female side, close to 80% of the mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs and where present show specific defects in the tapetum layer, reducing the number of functional pollen per anther by up to ~90%. Transcriptional profiling identified more than 600 down-regulated genes in ashh2 mutant inflorescences, including genes involved in determination of floral organ identity, embryo sac development and anther/pollen development. We observed a reduction of H3K36 trimethylation (me3) but not H3K4me3 or H3K36me2 in chromatin of such genes. Thus, our analysis strongly suggests that ASHH2 works via H3K36 trimethylation in the regulation of genes essential in reproductive development.